EP4272753A1 - Formulation containing soluble gp130 dimer and method for using same - Google Patents

Formulation containing soluble gp130 dimer and method for using same Download PDF

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Publication number
EP4272753A1
EP4272753A1 EP21914727.9A EP21914727A EP4272753A1 EP 4272753 A1 EP4272753 A1 EP 4272753A1 EP 21914727 A EP21914727 A EP 21914727A EP 4272753 A1 EP4272753 A1 EP 4272753A1
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EP
European Patent Office
Prior art keywords
aqueous formulation
formulation according
fusion protein
trehalose
colitis
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EP21914727.9A
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German (de)
English (en)
French (fr)
Inventor
Shuyun LU
Zheru ZHANG
Junhua QIAO
Jing Zhu
Jing Wang
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I Mab Biopharma Hangzhou Co Ltd
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I Mab Biopharma Hangzhou Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention belongs to the field of biopharmaceutical research, and specifically relates to a formulation containing a gp130 dimer and the use thereof for the treatment of various IL-6-mediated conditions including an inflammatory disease and cancer.
  • Glycoprotein 130 (also known as gp130, IL6ST, IL6-beta or CD130) is a transmembrane protein. It forms one subunit of a type I cytokine receptor within the IL-6 receptor family and is very important for signal transduction following cytokine engagement. Structurally, gp130 consists of five fibronectin type-III domains and one immunoglobulin-like C2-type domain in its extracellular portion.
  • IL-6 All members of the IL-6 receptor family form complexes with gp130 for signal transduction.
  • IL-6 binds to an IL-6 receptor, and then the complex formed by these two proteins is associated with gp130. After that, the complex consisting of the three proteins is homodimerized to form a hexameric complex which can generate downstream signals.
  • IL-6 is a pleiotropic cytokine generated by a hematopoietic cell and a non-hematopoietic cell, for example, in response to infections and tissue injuries.
  • IL-6 exerts its multiple biological activities by means of two main signal transduction pathways, including the so-called classic ligand-receptor pathway via a membrane-bound IL-6R mainly existing on hepatocytes and some leukocytes, and the trans-signal transduction pathway (the trans-signaling pathway) via a circulating sIL-6R (soluble IL-6R) derived from proteolytic cleavage of a membrane-bound IL-6R or derived from alternative splicing.
  • soluble IL-6R soluble IL-6R
  • the IL-6 directly binds to the membrane-bound IL-6R on the surfaces of a limited range of cell types.
  • the IL-6/IL-6R complex is associated with a dimer preformed by a signal-transducing gp130 receptor protein, which causes a gp130 homodimer to change spatially and thus triggers an intracellular signal transduction cascade.
  • Classic signal transduction is responsible for an acute inflammation defense mechanism and key physiological IL-6 functions, such as the growth and regeneration signals of enterocytes.
  • the extracellular domains of IL-6R and gp130 can be generated by translating an alternatively-spliced mRNA, and no membrane-anchored domains exist, thereby generating sIL-6R and gp130 variants.
  • the activity of the IL-6/sIL-6R complex is usually controlled by high-level sgpl30 (soluble gp130) existing in circulation, and this complex effectively competes with membrane-bound gp130.
  • the gp130 dimer in the present invention has a higher binding affinity in comparison with the natural sgpl30 and thus has a stronger ability to inhibit IL-6 signal transduction.
  • the formulation of the present invention enables the gp 130 dimer to be more stable during production, transportation and application.
  • the present invention describes a formulation containing a gp130 dimer (or "fusion protein containing gp130", or “fusion protein” for short), and the use thereof for the treatment of various IL-6-mediated conditions including an inflammatory disease and cancer.
  • the formulation comprises a histidine salt buffer system, has high stability at a pH of about 7.6, and can be safely administered to humans at various doses.
  • this description describes an aqueous formulation (also can be referred to as a liquid formulation) and a lyophilized formulation, comprising a fusion protein, a 20-30 mM histidine salt, 220-280 mM trehalose and 0.01(w/v)%-0.03(w/v)% polysorbate 80 and having a pH of 7.0-8.2, wherein the fusion protein comprises two monomers having amino acid sequences as set forth in SEQ ID NO: 1, and the two monomers are connected by a plurality of disulfide bonds.
  • the aqueous formulation comprises the fusion protein at a concentration of at least 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL or at least 30 mg/mL.
  • the aqueous formulation has a pH of 7.4-7.8. In some embodiments, the aqueous formulation has a pH of 7.6.
  • the aqueous formulation comprises a 24-26 mM histidine salt. In some embodiments, the aqueous formulation comprises a 25 mM histidine salt.
  • the aqueous formulation comprises 240-260 mM trehalose. In some embodiments, the aqueous formulation comprises 250 mM trehalose.
  • the aqueous formulation comprises 0.015(w/v)%-0.025(w/v)% polysorbate 80. In some embodiments, the aqueous formulation comprises 0.02(w/v)% polysorbate 80.
  • the aqueous formulation further comprises a tonicity agent (also known as an osmo-regulator or a stabilizer), a surfactant, an antioxidant, a preservative or a mixture thereof.
  • a tonicity agent also known as an osmo-regulator or a stabilizer
  • surfactant also known as an osmo-regulator or a stabilizer
  • antioxidant also known as an osmo-regulator or a stabilizer
  • preservative a mixture thereof.
  • each of the fusion protein molecules comprises no more than six galactose- ⁇ -1,3-galactose moieties. In some embodiments, each of the fusion protein molecules comprises no more than three, two or one galactose- ⁇ -1,3-galactose moiety.
  • the fusion protein comprises glycans, wherein on average at least 52% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average at least 54% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average 52%-65% of the glycans comprise one or more sialic acid residues.
  • the aqueous formulation comprises the fusion protein at a concentration of at least 25 mg/mL, a 24-26 mM histidine salt, 240-260 mM trehalose and 0.015(w/v)%-0.025(w/v)% polysorbate 80, with a pH of 7.6-7.8.
  • the aqueous formulation comprises the fusion protein at a concentration of 30 mg/mL, a 25 mM histidine salt, 250 mM trehalose and 0.02(w/v)% polysorbate 80, with a pH of 7.6.
  • the aqueous formulation comprises no additional amino acid salts at a concentration higher than 10 mM (or higher than 5 mM, 2 mM, 1 mM, 0.1 mM or 0.01 mM) other than the histidine salt, or preferably the aqueous formulation comprises no additional amino acid salts at all.
  • the aqueous formulation comprises no additional sugars at a concentration higher than 10 mM (or higher than 5 mM, 2 mM, 1 mM, 0.1 mM or 0.01 mM) other than the trehalose, or preferably the aqueous formulation comprises no additional sugars at all.
  • the aqueous formulation is used for the treatment of an inflammatory disease or an IL-6-mediated condition in a human.
  • the inflammatory disease or IL-6-mediated condition is an inflammatory bowel disease, preferably the treatment induces amelioration of the inflammatory bowel disease.
  • the inflammatory bowel disease is Crohn's disease or ulcerative colitis, preferably the treatment maintains amelioration of the inflammatory bowel disease.
  • the inflammatory disease or IL-6-mediated condition is rheumatoid arthritis, psoriasis, uveitis or atherosclerosis.
  • the inflammatory disease or IL-6-mediated condition is colitis unrelated to inflammatory bowel disease, preferably the colitis is radiation colitis, diverticular colitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis or colitis caused by an allergy affecting the colon.
  • This description also describes a dry formulation, which can be obtained by lyophilizing any of the aqueous formulations described herein or can generate any of the aqueous formulations described herein by adding water.
  • protein and polypeptide are used interchangeably and, in their broadest sense, refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, e.g., ester and ether.
  • the protein or peptide must comprise at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise the protein's or peptide's sequence.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs and peptidomimetics. Single-letter and three-letter abbreviations of naturally occurring amino acids are listed below.
  • composition is intended to refer to a combination of an active agent and another inert (e.g., a detectable reagent or label) or active compound or composition (e.g., an adjuvant).
  • pharmaceutical composition is intended to comprise a combination of an active agent with an inert or active carrier, making the composition suitable for diagnostic or therapeutic use in-vitro, in-vivo or ex-vivo.
  • aqueous formulation refers to a liquid formulation using water as a solvent.
  • the aqueous formulation is a formulation that does not require lyophilizing, spray-drying and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).
  • buffer represents a pharmaceutically acceptable excipient, which stabilizes the pH of a pharmaceutical formulation.
  • Suitable buffers are well known in the art and can be found in the literature.
  • Pharmaceutically acceptable buffers include, but are not limited to, a tris buffer, an arginine buffer, a histidine buffer, a citrate buffer, a succinate buffer and a phosphate buffer.
  • the pH can be adjusted with an acid or base known in the art, such as succinic acid, hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, citric acid, succinate, citrate, tris base, histidine, histidine HCl, sodium hydroxide and potassium hydroxide.
  • Suitable buffers include, but are not limited to, a histidine buffer, 2-morpholinoethanesulfonic acid (MES), dimethyl arsenate, phosphate, acetate, succinate and citrate.
  • concentration of the buffer may be about 4 mM to about 60 mM, or alternatively about 4 mM to about 40 mM, or alternatively about 5 mM to about 25 mM.
  • treatment refers to reversing, alleviating, delaying the onset of or inhibiting the progress of a disease or condition as described herein or one or more symptoms thereof.
  • treatment may be administered after one or more symptoms are developed.
  • treatment may be administered in the absence of symptoms.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms are relieved, for example, to prevent or delay their recurrence.
  • IM001 is a dimer containing two single-stranded gp130-Fc fusion proteins, and can be used for the treatment of various IL-6-mediated conditions including an inflammatory disease and cancer. Similar to other protein drugs, the solubility, stability and activity of IM001 are affected by the environment. Therefore, it would be not easy to develop a suitable formulation comprising a suitable buffer system.
  • the inventor prepares 12 kinds of pH/buffer system formulas (Table 2) and 9 kinds of excipient and surfactant (aqueous formulation) screening formulas (Table 7), investigates the stability thereof at 30°C for 2 weeks and compares these formulas by means of DSC, DLS, appearance, protein concentration, pH, SEC-HPLC and SDS (reducing/non-reducing) methods. It has been found that at pH ⁇ 6.5, visible particles are obviously generated and the protein is also very unstable, whereas the protein is more stable in a buffer system having a pH of 8.0 (slightly alkaline). In addition, with regard to a same buffer system having a pH of 8.0, the stability in the histidine buffer system is higher than that in the glycine and Tris buffer systems. Interestingly, adding IM001 to a buffer system may lead to pH drift, and after the protein is added to a histidine buffer system, the pH drifts from 7.6 to 8.0.
  • the inventor of the present application after investigating various aqueous formulation formulas, finds that the protein at a concentration of 15 mg/mL leads to the best stability under the protection of sucrose and polysorbate 80. Unfortunately, the stability is insufficient when the protein concentration reaches 30 mg/mL.
  • a stable aqueous formulation having a higher concentration can be easily developed from many proteins comprising Fc fragments, such as an antibody.
  • Fc fragments such as an antibody.
  • the inventor of the present application believes that because the fusion protein molecular has a characteristic of being easily unstable at high temperature or high concentration and isoelectric points set limitations on the selections of pH buffer systems, the development of the aqueous formulation is more challenging than the development of biologics molecular formulations such as common monoclonal antibodies.
  • the present application provides an aqueous formulation and lyophilized formulation suitable for IM001, comprising a fusion protein, a histidine salt, trehalose and polysorbate.
  • the aqueous formulation can form the lyophilized formulation after being lyophilized.
  • the lyophilized formulation can generate the aqueous formulation by adding an appropriate amount of water. Such an aqueous formulation may also be injected into a patient for the treatment of corresponding diseases.
  • the fusion protein (IM001) here comprises two monomers having amino acid sequences as set forth in SEQ ID NO: 1, and the two monomers are connected by a plurality of disulfide bonds.
  • each of the fusion protein molecules comprises no more than six galactose- ⁇ -1,3-galactose moieties.
  • each of the fusion protein molecules comprises no more than three, two or one galactose- ⁇ -1,3-galactose moiety.
  • the fusion protein comprises glycans, wherein on average at least 52% of the glycans comprise one or more sialic acid residues.
  • the fusion protein comprises glycans, wherein on average at least 54% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average 52%-65% of the glycans comprise one or more sialic acid residues.
  • the aqueous formulation comprises the fusion protein at a concentration of at least 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL or at least 30 mg/mL. In some embodiments, the aqueous formulation comprises the fusion protein at a concentration of 10-60 mg/mL, 15-45 mg/mL, 20-40 mg/mL, 25-35 mg/mL or 30 mg/mL.
  • the aqueous formulation comprises a histidine salt at a concentration of at least 10 mM. In some embodiments, the aqueous formulation comprises a histidine salt at a concentration of at least 15 mM, 20 mM, 25 mM, 30 mM or 35 mM. In some embodiments, the aqueous formulation comprises a 10-50 mM histidine salt, or a 10-40 mM, 15-35 mM, 20-30 mM, 22-28 mM or 24-26 mM histidine salt. In some embodiments, the aqueous formulation comprises a 25 mM histidine salt.
  • the aqueous formulation comprises trehalose at a concentration of at least 100 mM. In some embodiments, the aqueous formulation comprises trehalose at a concentration of at least 100 mM, 150 mM, 200 mM, 250 mM or 300 mM. In some embodiments, the aqueous formulation comprises 100-400 mM trehalose, or 150-350 mM, 200-300 mM, 220-280 mM, 240-260 mM or 245-255 mM trehalose. In some embodiments, the aqueous formulation comprises 250 mM trehalose.
  • the aqueous formulation comprises polysorbate, such as polysorbate 20 or polysorbate 80. In some embodiments, the aqueous formulation comprises polysorbate at a concentration of at least 0.005(w/v)%. In some embodiments, the aqueous formulation comprises polysorbate at a concentration of at least 0.01(w/v)%, at least 0.015(w/v)%, at least 0.02(w/v)%, or at least 0.025(w/v)%. In some embodiments, the aqueous formulation comprises 0.01(w/v)%-0.03(w/v)% polysorbate 80.
  • the aqueous formulation comprises 0.015(w/v)%-0.025(w/v)% polysorbate 80. In some embodiments, the aqueous formulation comprises 0.018(w/v)%-0.022(w/v)% polysorbate 80. In some embodiments, the aqueous formulation comprises 0.019(w/v)%-0.021(w/v)% polysorbate 80. In some embodiments, the aqueous formulation comprises 0.02(w/v)% polysorbate 80.
  • the aqueous formulation has a pH of 7.0 or higher. In some embodiments, the aqueous formulation has a pH of 7.1 or higher, 7.2 or higher, 7.3 or higher, 7.4 or higher, 7.5 or higher, or 7.6 or higher. In some embodiments, the aqueous formulation has a pH of 7.0-8.2, or 7.1-8.1, 7.2-8.0, 7.3-7.9, 7.4-7.8 or 7.5-7.7 or 7.55-7.65. In some embodiments, the aqueous formulation has a pH of 7.6.
  • the aqueous formulation comprises the fusion protein at a concentration of at least 25 mg/mL, a 24-26 mM histidine salt, 240-260 mM trehalose and 0.015(w/v)%-0.025(w/v)% polysorbate 80, with a pH of 7.4-7.8.
  • the aqueous formulation consists of the fusion protein at a concentration of at least 25 mg/mL, a 24-26 mM histidine salt, 240-260 mM trehalose and 0.015(w/v)%-0.025(w/v)% polysorbate 80, with a pH of 7.4-7.8.
  • the aqueous formulation comprises the fusion protein at a concentration of at least 25 mg/mL, a 25 mM histidine salt, 250 mM trehalose and 0.02(w/v)% polysorbate 80, with a pH of 7.6.
  • the aqueous formulation consists of the fusion protein at a concentration of 30 mg/mL, a 25 mM histidine salt, 250 mM trehalose and 0.02(w/v)% polysorbate 80, with a pH of 7.6.
  • the aqueous formulation further comprises a tonicity agent (also called an osmo-regulator or a stabilizer), a surfactant, an antioxidant, a preservative or a mixture thereof.
  • a tonicity agent also called an osmo-regulator or a stabilizer
  • surfactant also called an antioxidant, a preservative or a mixture thereof.
  • the aqueous formulation further comprises a tonicity agent (also called an osmo-regulator or a stabilizer).
  • a tonicity agent also called an osmo-regulator or a stabilizer.
  • the term "tonicity agent” represents a pharmaceutically acceptable agent for regulating the tonicity of the formulation. Isotonicity generally relates to an osmotic pressure relative to a solution, usually relative to a solution of human serum.
  • the formulation may be hypotonic, isotonic or hypertonic. In one aspect, the formulation is isotonic.
  • the isotonic formulation is a liquid, or a liquid reconstituted from a solid form (e.g., from a lyophilized form) and represents a solution which has the same tonicity as some other solutions (e.g., a physiological salt solution and serum) for comparison.
  • Suitable isotonic agents include, but are not limited to, sodium chloride, potassium chloride, glycerol, mannitol and any component from amino acids and sugars as defined herein, and a combination thereof.
  • the aqueous formulation further comprises a surfactant.
  • surfactant refers to a pharmaceutically acceptable organic substance having an amphipathic structure, that is, the surfactant consists of groups with opposite solubility trends, generally oil-soluble hydrocarbon chains and water-soluble ionic groups.
  • Surfactants can be classified, depending on the charge of the surface-active moiety, into anionic surfactants, cationic surfactants and nonionic surfactants.
  • Surfactants are usually used as a wetting agent, an emulsifier, a solubilizer and a dispersant for various pharmaceutical compositions and biomaterial formulations.
  • the amount of the surfactant is described as a percentage (w/v%) expressed in the weight/volume percent.
  • Suitable pharmaceutically acceptable surfactants include, but are not limited to, a group consisting of polyoxyethylene sorbitan fatty acid ester (Tween), polyoxyethylene alkyl ether, alkyl phenyl polyoxyethylene ether (Triton-X), a polyoxyethylene-polyoxypropylene copolymer (poloxamer, Pluronic) or sodium dodecyl sulfate (SDS).
  • the polyoxyethylene sorbitan fatty acid ester comprises polysorbate 20 (sold under the trademark Tween 20 TM ) and polysorbate 80 (sold under the trademark Tween 80 TM ).
  • the polyethylene-polypropylene copolymer comprises those sold under the name Pliironic ® F68 or poloxamer 188 TM .
  • the polyoxyethylene alkyl ether comprises those sold under the trademark Bri j TM .
  • the alkyl phenyl polyoxyethylene ether comprises those sold under the trade name Triton-X.
  • the aqueous formulation further comprises an antioxidant.
  • the "antioxidant” refers to a molecule capable of slowing down or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidant. Oxidation reactions can produce free radicals, which start chain reactions that make a protein therapeutic agent instable and eventually affect the activity of a product. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibits other oxidation reactions by being oxidized themselves. Therefore, antioxidants are usually reducing agents, chelating agents and oxygen scavengers, such as citrate, EDTA, DPTA, mercaptan, ascorbic acid or polyphenol.
  • Nonlimiting examples of antioxidants comprise ascorbic acid (AA, E300), thiosulfate, methionine, tocopherol (E306), propyl gallate (PG, E310), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA, E320) and butylated hydroxytoluene (BHT, E321).
  • the aqueous formulation further comprises a preservative.
  • the "preservative" is a natural or synthetic chemical that is added to products such as foods, pharmaceuticals, paints, biological samples and wood to prevent decomposition resulting from microbial growth or undesirable chemical changes.
  • the preservative additive can be used alone or in combination with other preservation methods.
  • the preservative may be an antimicrobial preservative which inhibits the growth of bacteria and fungi, or an antioxidant such as an oxygen scavenger which inhibits the oxidation of components.
  • Common antimicrobial preservatives comprise benzalkonium chloride, benzoic acid, cholorohexidine, glycerin, benzoic acid, potassium sorbate, thimerosal, sulfite (sulfur dioxide, sodium bisulfite, potassium bisulfite, etc.) and disodium EDTA.
  • Other preservatives comprise those commonly used for parenteral proteins, such as benzyl alcohol, phenol, m-cresol, chlorobutanol or methylparaben.
  • some commonly used excipients of biologics formulations such as glycine, Tris and mannitol, do not contribute to the formation of an excellent aqueous formulation or lyophilized formulation. Therefore, in some embodiments, the aqueous formulation does not contain these excipients.
  • the aqueous formulation comprises no additional amino acid salts at a concentration higher than 10 mM (or higher than 5 mM, 2 mM, 1 mM, 0.1 mM or 0.01 mM), such as arginine, lysine, asparagine, glutamine, glycine or salts thereof, other than the histidine salt.
  • the aqueous formulation comprises no additional amino acid salts, such as arginine, lysine, asparagine, glutamine, glycine or salts thereof, other than the histidine salt.
  • the aqueous formulation comprises no additional sugars at a concentration higher than 10 mM (or higher than 5 mM, 2 mM, 1 mM, 0.1 mM or 0.01 mM), such as sucrose, other than the trehalose. In some embodiments, the aqueous formulation comprises no additional sugars, such as sucrose, other than the trehalose.
  • this description also describes a corresponding dry formulation, such as a lyophilized formulation.
  • the dry formulation can be obtained by lyophilizing the aqueous formulation described in this description.
  • the dry formulation can generate the aqueous formulation described in this description by adding an appropriate amount of water.
  • the formulation described in this description can be used for the treatment of various corresponding diseases.
  • the gp130 dimer in the present invention has a higher binding affinity in comparison with the natural soluble gpl30 and thus has a stronger ability to inhibit IL-6 signal transduction.
  • IL-6 signal transduction is related to many diseases, comprising diseases briefly described below and generally understood by those skilled in the art.
  • Chronic inflammation such as Crohn's disease (CD), ulcerative colitis (UC), rheumatoid arthritis (RA) or psoriasis is histologically related to the presence of mononuclear cells such as macrophages and lymphocytes, which persist in tissues after having been acquired for the resolution of the acute inflammatory phase.
  • IL-6 seems to have detrimental role favoring mononuclear cell accumulation at the site of injury by inducing sustained MCP-1 secretion, angioproliferation and antiapoptotic functions on T cells.
  • IBD Inflammatory bowel disease
  • CD or UC is a chronic inflammation occurring in the intestinal tract of a susceptible individual, and is believed to be unrelated to a specific pathogen.
  • the uncontrolled activation of mucosal CD4+T-lymphocytes accompanied by continuous excessive release of proinflammatory cytokines, induces pathogenic gastrointestinal inflammations and tissue injuries.
  • the main activated immune cells involved in the pathogenesis of IBD are intestinal T cells and macrophages.
  • IL-6 is shown as a central cytokine in IBD in the human body. It has been found that (3) and UC patients generate increased levels of IL-6 in comparison with the control group, and the level of the IL-6 is related to clinical activities. It has also been found that the level of SIL-6R in (3) patients is increased, and accordingly, the level of the IL-6/sIL-6R complex in serum is increased. Lamina limbal mononuclear cells obtained from surgical colon specimens from CD and UC patients show that both CD4+T cells and macrophages generate increased amounts of IL-6 in comparison with the control group.
  • SIL-6R is released via shedding from the surfaces of macrophages and mononuclear cells, which is accompanied by increased production related to the increased level of IL-6.
  • mucosal T cells show strong evidence for IL-6 trans-signal transduction, which is accompanied by activation of STAT3, bcl-2 and bcl-xl.
  • the blockade of the IL-6 trans-signal transduction causes T cell apoptosis, which indicates that the IL-6/sIL-6R system mediates the resistance of T cells to apoptosis in CD.
  • the formulation of the present invention can be used to treat an IL-6-mediated condition.
  • the IL-6-mediated condition comprises an inflammatory disease or cancer.
  • the polypeptide and composition described herein may be administered to a subject with an inflammatory disease, such as juvenile idiopathic arthritis, Crohn's disease, colitis (e.g., colitis unrelated to IBD, including radiation colitis, diverticular colitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis or colitis resulting from an allergy affecting the colon), dermatitis, psoriasis, uveitis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, nephritis, Parkinson's disease, ulcerative colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma and various cardiovascular diseases such as atherosclerosis and vasculitis.
  • the inflammatory disease such
  • the inflammatory disease or IL-6-mediated condition is an inflammatory bowel disease, preferably wherein the treatment induces amelioration of the inflammatory bowel disease.
  • the inflammatory bowel disease is Crohn's disease or ulcerative colitis, preferably wherein the treatment maintains amelioration of the inflammatory bowel disease.
  • the inflammatory disease or IL-6-mediated condition is rheumatoid arthritis, psoriasis, uveitis or atherosclerosis.
  • the inflammatory disease or IL-6-mediated condition is colitis unrelated to inflammatory bowel disease, preferably wherein the colitis is radiation colitis, diverticular colitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis or colitis resulting from an allergy affecting the colon.
  • the inflammatory disease or IL-6-mediated condition is selected from Crohn's disease, ulcerative colitis, rheumatoid arthritis and psoriasis, more preferably from Crohn's disease and ulcerative colitis.
  • the treatment can comprise amelioration of the condition, maintenance of the amelioration of the condition, or both.
  • cancers which include, but are not limited to, multiple myeloma, plasma cell leukemia, renal cell carcinoma, Kaposi's sarcoma, colorectal cancer, gastric cancer, melanoma, leukemia, lymphoma, glioma, glioblastoma multiforme, lung cancer (including, but not limited to non-small cell lung cancer (NSCLC; adenocarcinoma and squamous cell carcinoma)), non-Hodgkin's lymphoma, Hodgkin's disease, plasmacytoma, sarcoma, thymoma, breast cancer, prostate cancer, hepatocellular carcinoma, bladder cancer, uterine cancer, pancreatic cancer, esophageal cancer, brain cancer, head and neck cancers, ovarian cancer, cervical cancer, testicular cancer, stomach cancer, esophageal cancer, liver cancer, acute lymphoblastic leukemia
  • a disease which is selected from a group consisting of sepsis, bone resorption (osteoporosis), cachexia, cancer-related fatigue, psoriasis, systemic juvenile idiopathic arthritis, systemic lupus erythematosus (SLE), mesangial proliferative glomerulonephritis, hypergammaglobulinemia, Castleman's disease, IgM gammopathy, cardiac myxoma and autoimmune insulin-dependent diabetes mellitus.
  • a disease which is selected from a group consisting of sepsis, bone resorption (osteorosis), cachexia, cancer-related fatigue, psoriasis, systemic juvenile idiopathic arthritis, systemic lupus erythematosus (SLE), mesangial proliferative glomerulonephritis, hypergammaglobulinemia, Castleman's disease, IgM gammopathy, cardiac myxoma
  • the present disclosure provides a way for producing the formulation.
  • the cDNA encoding SEQ ID NO: 1 may be cloned into a vector such that the signal peptide is linked in-frame to an amino terminus of the amino acid sequence of an antibody chain.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the design of the expression vector comprising the selection of regulatory sequences, may depend on factors such as the selection of host cells to be transformed and the expression level of desired proteins.
  • Regulatory sequences for the expression of mammalian host cells comprises viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as a CMV promoter/enhancer), simian virus 40 (SV40) (such as an SV40 promoter/enhancer), adenovirus (such as an adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters (such as natural immunoglobulin and actin promoters).
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • AdMLP adenovirus major late promoter
  • polyoma and strong mammalian promoters (such as natural immunoglobulin and actin promote
  • the host cell may be a mammalian cell, an insect cell, a plant cell, a bacterial cell or a yeast cell, preferably the cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.
  • CHO cells are (CHO)/dhfr - cells obtained from the European Collection of Authenticated Cell Cultures (ECACC, No. 9406067).
  • the host cell is a CHO cell, and the nucleic acid encoding the polypeptide is codon-optimized for use in the CHO cell.
  • the present disclosure comprises a fusion protein produced by the method disclosed herein.
  • the dimer has the characteristics described herein (e.g., % galactose- ⁇ -1,3-galactose moieties per mole of polypeptide, sialylation).
  • the dimer generated by the method can be used for the preparation of a suitable composition.
  • the fusion protein molecule produced in this way comprises no more than six galactose- ⁇ -1,3-galactose moieties, or no more than three, two or one galactose- ⁇ -1,3-galactose moiety.
  • the fusion protein molecule produced in this way comprises glycans, wherein on average at least 52% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average at least 54% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average 52%-65% of the glycans comprise one or more sialic acid residues.
  • the IM001 was obtained from a CHO-K1 engineered cell expressing a gp130-Fc fusion protein gene by means of cell culture, separation and purification.
  • Table 1 Amino acid sequence of a single strand of IM001 Single strand of IM001 (SEQ ID NO: 1)
  • the cDNA sequence of the IM001 was expressed in a CHO cell expression system.
  • the presence of the IgGl Cys-Pro-Pro-Cys sequence in the Fc region resulted in the dimerization of two identical gpl30-Fc subunits by means of sulfhydryl residues on the Fc region, which together form the IM001.
  • the pilot manufacturing and purification process of the IM001 drug substance was as follows. Before inoculated into a bioreactor for production, cells from a WCB vial were revived and gradually expanded using a protein-free culture medium. Upon completion of the cell culture, cells and cell debris were removed by filtration of the culture. Purification consisted of three column chromatography steps, one concentration and diafiltration step and two specific viral clearance steps (viral inactivation treatment and nanofiltration, and removal of enveloped and non-enveloped viruses). Following concentration and diafiltration, an excipient was added to formulate a drug substance. The prepared IM001 was filtered into a container by means of a 0.22- ⁇ M filter membrane.
  • This example also attempted to screen the most stable pH/buffer system for the IM001.
  • the IM001 stock solutions were exchanged into 20 mM acetate (pH 4.5, pH 5.0 and pH 5.5), citrate (pH 5.0), histidine-aspartate (pH 5.0 and pH 5.5), histidine (pH 5.5, pH 6.0, pH 6.5, pH 7.0 and pH 8.0) and phosphate (pH 7.0) by means of dialysis.
  • concentration of the protein was adjusted to about 30 mg/mL, and then the protein was filtered with a 0.22- ⁇ m PVDF membrane filter. Following filtration, the samples were bottled and sealed. All manipulations took place in a biological safety shield.
  • the sample in one of the bottles was used for T0, and appropriate stability investigation conditions were selected based on the DSC and HT-DLS results of T0.
  • the rest samples were stored under the conditions, and each bottle was subjected to sampling analysis at time points specified in Table 2.
  • the reducing SDS-PAGE results showed that after storage at 30°C for 2 weeks, there was no decrease in purities of p10 (His at pH 7.0), p11 (His at pH 8.0) and p12 (Phosphate at pH 7.0), while there showed different degrees of decrease in purities of the rest samples, with decrease ranges of 10.8%-50.0%.
  • the non-reducing SDS-PAGE results showed that there were different degrees of decrease in purities of all samples, wherein the decrease ranges of p10 (His at pH 7.0), p11 (His at pH 8.0) and p12 (Phosphate at pH 7.0) were less than those of the rest samples which were 14.6%-65.7%. Table 6.
  • SDS-PAGE results of IM001 pH/buffer system screening NO.
  • SDS-PAGE R
  • SDS-PAGE NR
  • Purity %) Main Band (kDa) Purity (%) (H + L) Main Band (kDa) T0 30C-1W 30C-2W T0 30C-1W 30C-2W T0 30C-1W 30C-2W T0 30C-1W 30C-2W 1 98.1 67.5 48.1 109 104 107 91.3 47.8 25.6 316 262 265 2 98.4 79.9 64.5 106 102 107 92.7 59.6 41.8 297 252 255 3 99.3 85.7 75.4 106 100 107 94.2 69.6 52.9 301 246 245 4 98.6 79.2 68.1 106 100 108 93.4 61.4 42.6 294 252 240 5 99.3 83.6 73.8 105 98 109 95.4 66.7 49.1 297 262 236 6 99.3 89.
  • the most stable pH/buffer system for the IM001 was screened by means of DSC, DLS, appearance, protein concentration, pH, SEC-HPLC and SDS-PAGE (reducing/non-reducing).
  • the DSC and DLS results showed that the Tm Onset and Tagg Onset of the IM001 molecule showed an increasing trend with the increase of pH, wherein the Tm Onset and Tagg Onset of p11 (His at pH 8.0) were higher than those of the rest samples.
  • the study purpose of this example was to screen an appropriate excipient stabilizer on the basis of pH/buffer system screening.
  • a 25 mM histidine buffer system at pH 8.0 was used as a main buffer system for the assessment of excipient screening.
  • the glycine buffer system at pH 7.5 and the Tris buffer system at pH 7.5 were added for investigation.
  • the IM001 stock solution was exchanged into a 25 mM histidine buffer system (pH 8.0), a 25 mM glycine buffer system (pH 7.5) and a 25 mM Tris buffer system (pH 7.5) by means of ultrafiltration centrifugation.
  • Formula Type of stability T0 Acceleration Long term Freezing and thawing Influence factor Vibration and shaking Preservation condition 25°C ⁇ 2°C 2°C to 8°C -70°C to RT 30°C ⁇ 2°C 300 rpm to 25°C Sampling point 2W 1M 3M 1M 3M 6M 12M 5C 1W 2W 7D F1 30 mg/mL 25 mM His pH 8.0, 250 mM Sucrose, 0.01% PS20 Y Y, z Y, Z Y Y, Z Y, Z Y, Z Y. Z Y Y.
  • Z Y, Z F2 30 mg/mL 25 mM His pH 8.0, 250 mM Sucrose, 0.02% PS80 Y Y, Z Y, Z Y Y, Z Y, G Y; Z Y. Z Y Y, Z Y, Z F3 30 mg/mL 25 mM His pH 8.0, 250 mM Trehalose.
  • the insoluble microparticle detection (MFI) results of the excipient and surfactant screening experiments were summarized in Table 11.
  • the freezing and thawing sample data showed that the number of insoluble microparticles in the F1 sample, which was frozen and thawed, was greater than those of the rest samples; after the samples were placed at 30°C for 2 weeks, the data showed that the number of insoluble microparticles in the F1 sample was greater than that of the rest samples, and the F4 sample was not subjected to an MFI test due to the presence of many visible particles; and after the samples were placed at 25°C for 1 month, the data showed that the number of insoluble microparticles in the F1 and F4 samples was higher than that of the rest samples.
  • the SEC-HPLC test results were summarized in Table 12. After the samples were placed at 25°C for 1 month and at 30°C for 2 weeks, the data showed that the decrease ranges of the F4 and F5 samples were both greater than those of the rest samples, the decrease range of F9 was smaller than those of the rest samples, and the decrease ranges of the rest samples were similar. After the samples were placed at 2°C to 8°C for 1 month, the data showed that the main-peak purity of the F5 sample was decreased by 2.1%, and the main-peak purities of the rest formula samples had no significant change. After the samples were freezed and thawed for 5 cycles, the data showed that the main-peak purity of the F8 sample was decreased by 10.3%, and the main-peak purities of the rest samples had no significant change.
  • the SDS-PAGE data were summarized in Table 13.
  • the reducing SDS-PAGE data showed that the purities of different samples showed no significant difference.
  • the non-reducing SDS-PAGE data showed that the purity of the F9 sample was superior to that of the rest samples, and the purity data of the rest samples were similar.
  • Table 13 SDS-PAGE results of excipient and surfactant screening experiments Formula no.
  • the data for F2 and F3 samples were similar under each investigation condition, indicating that sucrose and trehalose had similar stabilizing effects on the protein; the main-peak purities of the F4 and F5 samples under each investigation condition were slightly lower than those of the rest samples, indicating that the histidine buffer system at pH 8.0 was superior to the glycine and Tris buffer systems; the data for F2 and F6 samples were similar under each investigation condition, indicating that F6 in which I mM methionine was added and F2 in which methionine was not added showed no significant difference; and the data for F2 and F7 samples were similar under each investigation condition, indicating that the stability of F7, which comprises a combination of 4% mannitol and 2% sucrose was similar to that of F2, which comprises 250 mM sucrose.
  • the main-peak purity of the F9 sample was higher than that of the F2 under each investigation condition, indicating that the aqueous formulation with 15 mg/mL proteins was more conducive to the stability of the IM001 in comparison with the aqueous formulation with 30 mg/mL proteins.
  • the reducing SDS-PAGE data showed that the purity data of different samples showed no significant difference.
  • the non-reducing SDS-PAGE data showed that the purity of the F9 sample was superior to that of the rest samples, and the purity data of the rest samples were similar.
  • F2 and F9 had the same composition except for different concentrations, indicating that the aqueous formulation with 15 mg/mL proteins was more conducive to the stability of the IM001 in comparison with the aqueous formulation with 30 mg/mL proteins.
  • the study purpose of this example was to screen the formula of a high-concentration lyophilized formulation.
  • the concentration of the IM001 was increased to 30 mg/mL, and the purity was decreased obviously with the prolonged time of storage at 25°C or higher temperature. Therefore, the formula of a lyophilized formulation was designed. See Table 14 for the scheme.
  • the IM001 stock solution was exchanged into a 25 mM histidine buffer system (pH 8.0); different excipient mother solutions and surfactant mother solutions were added respectively according to the experimental scheme; and the protein content was adjusted to 30 mg/mL by adding different kinds of sugar mother solutions and surfactant mother solutions, respectively.
  • Tg' glass-transition temperature
  • Tc lyophilizing collapse temperature
  • insoluble microparticle detection (MFI) results of the reconstituted solutions of the lyophilized products were summarized in Table 20.
  • the number of insoluble microparticles in different formulas had no obvious differences, and in comparison with the T0 sample, the number of insoluble microparticles in the samples placed at 25°C for 1 month or 40°C for 2 months was not increased obviously after reconstituting.
  • Table 20 Insoluble microparticle detection (MFI) results of reconstituted solutions of lyophilized products Formula no. Sample no.
  • the insoluble microparticles in the 25C-3M, 40C-1M and 40C-2M samples of F2 were obviously fewer than those in the T0 sample, and with regard to F15, it can also be found that the number of insoluble microparticles in the 40C-1M and 40C-2M samples was obviously lower than that in the 25C-3M sample.
  • the SEC-HPLC test results were summarized in Table 21. After all the formulas were placed under acceleration conditions (25°C) and high temperature conditions (40°C) for 2 weeks to 3 months, except that the SEC purity of F7 was lightly decreased, the SEC purities of the rest samples were generally unchanged, indicating that the stability of the lyophilized product was significantly improved in comparison with the liquid formulation.
  • the stability of F7 (comprising 4% mannitol and 2% sucrose) was worse than that of the rest formulas, which may be related to the crystallization of mannitol after annealing and the loss of protective effects of sugar on the protein.
  • the rest formulas had generally unchanged SEC main peak contents and all had good stability.
  • the 5 lyophilized products investigated all had good appearances, and were white, loose blocks.
  • the moisture content determination results were 1.1% to 1.6%, all less than 3%, so the moisture contents were qualified.
  • the reconstituted solutions were all slightly yellow, slightly opalescent and free of visible particles after the products were placed under acceleration conditions (25°C) and high temperature conditions (40°C) for 2 weeks to 3 months.
  • the Tg' of F7 (comprising 4% mannitol and 2% sucrose) was -33.2°C, and the Tg' of the rest formulas was about -26°C to -27°C. MFI results of different formulas showed no obvious differences.
  • the buffer system screening results show that the His buffer salt system at pH 8.0 (formula: pH 7.6) has the best stability.
  • the excipient and surfactant screening results show that the stability in the histidine buffer system at pH 8.0 is stronger than that in the glycine and Tris buffer systems; the protective effects of sucrose and trehalose are similar and both superior to that of mannitol; the system with a protein concentration of 15 mg/mL has a more superior stability than the system with a protein concentration of 30 mg/mL; and the aqueous formulation with F9 (15 mg/mL protein, 25 mM His, 250 mM Sucrose, and 0.02% PS80, pH 7.6) has the best stability.
  • the lyophilized formulation screening experiment results show that the lyophilized products always have an obviously improved stability in comparison with the solution formula, wherein F7 (30 mg/mL protein, 25 mM His pH 8.0, 4% Mannitol, 2% Sucrose and 0.02% PS80) shows slightly poorer performance, whereas the rest formulas all show excellent stability.
  • F15 (30 mg/mL protein, 25 mM His, 250 mM Trehalose, and 0.02% PS80, pH 7.6) is selected as the formula of the lyophilized formulation of the IM001.

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